Combined cgh &amp; allele specific hybridisation method

ABSTRACT

The invention combines the fields of comparative genomic hybridisation (CGH) analysis and SNP array analysis. It relates to methods for detecting and mapping genetic abnormalities associated with various diseases. In particular the invention provides a method for simultaneously performing array CGH and SNP array analysis on a genomic DNA sample comprising contacting a nucleic acid array which comprises a first probe set and a second probe set with a genomic DNA sample, comprising a test and reference sample, under hybridisation conditions, comparing the amount of test sample and reference sample hybridised to the hybridisation probes of the first probe set, comparing the amount of test sample and reference sample hybridised to the hybridisation probes of the second probe set; and using the data obtained to determine the copy number of at least one locus; and at least one SNP in the genomic DNA sample.

This application claims the benefit of United Kingdom patentapplications 1000315.0 (filed 8 Jan. 2010), 1006438.4 (filed 16 Apr.2010) and 1014226.3 (filed 25 Aug. 2010), the complete contents of allof which are hereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

The invention combines the fields of comparative genomic hybridisation(CGH) analysis and SNP (or other sequence variation) array analysis. Itrelates to methods for detecting and mapping genetic abnormalitiesassociated with various diseases. It relates to the use of nucleic acidhybridisation methods for simultaneously detecting copy number and SNP(or other sequence variation) information in genomic DNA samples.

BACKGROUND ART

Comparative genomic hybridisation (CGH) is a molecular-cytogeneticmethod for the analysis of copy number changes (gains/losses) in asubject's DNA. The technique relies on the comparison of two labelledsamples by allowing them to hybridise and subsequently looking forregions of differential hybridisation. It has been used particularly incytogenetic analysis, where it allows the comparison of a genomeisolated from a clinical (test) sample (e.g. derived from a cancerpatient) with a control (reference) sample in a single hybridisation. Itwas originally disclosed in reference 1.

Whereas early CGH methods relied on hybridisation to a referencechromosome sample, array-based CGH methods have since been developed[2-4]. In these methods, the reference chromosome is replaced by anarray of immobilised nucleic acid probes, with the individualimmobilised sequences having known chromosomal locations and coveringthe genome to a desired degree. By choosing appropriate probes, thismethod gives the potential to cover any genomic region of interest, andto any desired resolution. Commercial array CGH kits are now available,including Spectral Chip from Perkin Elmer, CytoChip from BlueGnome, andCGH products from Nimblegen.

These two distinct methods are referred to as ‘chromosomal CGH’ and‘array CGH’.

Copy number variation in humans can result in certain disease types and,although CGH is a powerful tool for analsing copy number changes in agiven subject's DNA, it is only able to provide information aboutunbalanced chromosomal changes. Structural chromosome aberrations suchas balanced reciprocal translocations or inversions can not be detected,as they do not change the copy number, nor is it able to detect copynumber neutral loss of heterozygosity (LOH) due to uniparental disomy(UPD).

UPD occurs when two copies of a chromosome, or part of a chromosome, areinherited from one parent and no copies from the other parent [5]. Whenthe (two) homologous chromosomes are inherited from one parent, this iscalled a heterodisomic UPD. Heterodisomy indicates a meiosis 1 error.When the two (identical) replica copies of a single homolog of achromosome are inherited, this is called an isodisomic UPD. Isodisomyindicates either a meiosis II error or postzygotic duplication.

Most occurrences of UPD result in no phenotypical anomalies. However,isodisomy can lead to the manifestation of rare recessive disorders, forexample Silver-Russell or Prader Willi syndromes.

Determining if UPD has occurred can use single-nucleotide polymorphisms(SNPs) as markers to track the chromosome and determine if LOH hasoccurred. A SNP is a DNA sequence variation occurring when a singlenucleotide—A, T, C, or G—in the genome differs between members of apopulation. For example, two sequenced DNA fragments from differentindividuals, AAGCCTA to AAGCTTA, contain a difference in a singlenucleotide at a given position. In this case there are two alleles: Cand T. Almost all common SNPs have only two variants. Within apopulation, SNPs can be assigned an allele frequency which indicates thepercentage of the population which possess a particular nucleotideresidue at the SNP position.

Standard arrays for array CGH are not capable of detecting SNPs. ArrayCGH typically uses longer oligos and therefore tolerates a higherstringency to achieve an optimal copy number variation result. SNParrays tend to use shorter oligos under lower stringency and thereforethe standard SNP arrays can, in theory, be used to obtain copy numbervariation (CNV) data, but because they are not typically capable ofgenerating data of a comparable quality to array CGH they are not usedfor this purpose. Thus, for example, reference 6 used two differentarrays for its combined array-CGH and SNP-LOH analysis.

Reference 7 discloses arrays which can be used for both array CGH andSNP analysis. SNPs are detected by allele-specific chain extension ofhybridised probes.

It is an object of the invention to provide methods and apparatuses forsimultaneous array CGH and SNP array analysis. In particular it is anobject of the invention to provide improved ways of distinguishing ifLOH at a locus is caused by deletion or isodisomy.

DISCLOSURE OF THE INVENTION

The invention is based on the combination of standard array CGHtechniques with SNP (or other sequence variation) array analysis.Despite the difference in hybridisation conditions used for CGH and SNParrays, the inventors have surprisingly found that CGH and SNP (andother sequence variation) analysis can be performed simultaneously onthe same nucleic acid array. Appropriate design and confirmatoryempirical screening can provide SNP probes that function underconditions used for standard array CGH, and guidance for the selectionof suitable probes is provided herein.

To design a SNP probe for use in the invention, the genomic location andgenotype details are obtained from a source such as EMBL or dbSNP(http://www.ebi.ac.uk/embl/ andhttp://www.ncbi.nlm.nih.gov/projects/SNP/). These data are then filteredfor SNPs which exhibit allelic frequencies in the range of 0.4 to 0.6 inorder to maximise the number of informative data points represented onthe array.

Fifty nucleotides of upstream and downstream flanking sequence from thegenome form part of the SNP probe region. Once added the probes areexamined for genome wide uniqueness using the BLAST and/or BLATalgorithms. Oligonucleotide probes of a defined length (e.g. sixtynucleotides in length) specific for these SNPs are generated bycombining genomic sequence with linker sequences ranging from 0 to 30nucleotides in length.

The position of the SNP in relation to the remainder of the genomicsequence of the probe is experimentally evaluated and the bestperforming versions selected for the final design.

In order to focus the effort of the experimental optimisation all SNPsare filtered for allele frequencies, homology scores of the genomicsequences and melting temperature of the probe sequences. Moreoverprobes representing all 4 possible alleles on both strands of thegenomic sequence are evaluated for every single selected SNP.

Accordingly, the invention provides a method for simultaneouslyperforming array CGH and SNP array analysis on a genomic DNA samplecomprising: (a) contacting a nucleic acid array which comprises a firstprobe set and a second probe set with a genomic DNA sample, comprising atest and reference sample, under hybridisation conditions, wherein: (i)the first probe set, for the detection of copy number variation by arrayCGH, comprises a plurality of hybridisation probes substantiallycomplementary to a plurality of target nucleotide sequences in thenucleic acid sample; and (ii) the second probe set comprises one or morepair(s) of hybridisation probes for a SNP position, wherein the pair(s)of probes differ in sequence such that a nucleic acid target present inthe sample can differentially hybridise to the two probes depending onthe nucleotide at the SNP position, and a probe's nucleotide at the SNPposition is not the 3′ terminal nucleotide; (b) comparing the amount oftest sample and reference sample hybridised to the hybridisation probesof the first probe set; (c) comparing the amount of test sample andreference sample hybridised to the hybridisation probes of the secondprobe set; and (d) using the data obtained in steps (b) and (c) todetermine the copy number of at least one locus and at least one SNP inthe genomic DNA sample.

The invention also provides a method for distinguishing if loss ofheterozygosity (LOH) at a locus is caused by chromosomal deletion orisodisomy comprising: (i) simultaneously performing array CGH and SNParray analysis on a genomic DNA sample according to the method of theinvention: (ii) using the data obtained from step (d) of part (i) todistinguishing if loss of heterozygosity (LOH) at a locus is caused bychromosomal deletion or isodisomy; wherein, if all of the SNPs locatedon a particular chromosome or region of a chromosome are identified ashomozygous and there is no indication of copy number variation in thesame region, then it is likely that the LOH is a consequence ofuniparental isodisomy; if the SNPs located in a particular chromosome orregion of a chromosome are not all homozygous and there is an indicationof copy number variation in that region, then it is likely that the LOHis a consequence of the chromosomal deletion and not from UPD.

The invention also provides a nucleic acid array that, when contactedwith a genomic DNA sample under hybridisation conditions, can (i)provide information about the sample relating to the copy number of oneor more loci in the genome; and (ii) distinguish between differentalleles present at one or more SNP positions in the genome.

Preferably the nucleic acid array comprises a first probe set and asecond probe set, wherein (i) the first probe set, for the detection ofcopy number variation by array CGH, comprises a plurality ofhybridisation probes substantially complementary to a plurality oftarget nucleotide sequences in the genomic DNA sample; and (ii) thesecond probe set comprises one or more pair(s) of hybridisation probesfor a SNP position, wherein the pair(s) of probes differ in sequencesuch that a nucleic acid target present in the sample can differentiallyhybridise to the two probes depending on the nucleotide at the SNPposition, and a probe's nucleotide at the SNP position is not the 3′terminal nucleotide.

The invention also allows for the detection of heterodisomy as describedbelow.

Preferably the method comprises detecting the amount of the nucleic acidsample bound to the first and second probe set.

Preferably the DNA sample is a human genomic DNA sample.

Genomic DNA Sample

The DNA sample to be analysed is a genomic DNA (gDNA) sample. Analysiswill generally be performed on total genomic DNA which, in a eukaryote,includes DNA from the nucleus and other organelles e.g. from themitochondria.

The invention can be used for comparing all types of DNA, and isparticularly suitable for analsing human cells, including cancer cells.

The genomic DNA sample includes DNA from both a test sample (the samplefor which information is to be determined) and a reference sample (asample of known content). DNA in the test sample and the referencesample may be labelled with first and second labels, respectively. Thefirst and second labels should be distinguishable from each other, e.g.they may be different colors such as green, red, blue, etc. Preferablythe labels are fluorescent dyes.

Samples for use with the present invention may be prepared using methodsknown in the art for preparing samples to be analysed by array CGH.Examples of such methods are well known in the art e.g. as known for theSpectral Chip and CytoChip products.

Because the starting material for array CGH procedure is genomic DNA,which is composed of long chromosomal DNA molecules, the sample DNAneeds to be shortened before being applied to the array. This isgenerally achieved by fragmenting the sample DNA. The sample DNA may befragmented using any suitable method, including but not limited torestriction digestion after amplification or sonication before randomprime labelling and amplification. Fragmentation of genomic DNA in asample can be achieved physically, chemically, or enzymatically.Physical and chemical fragmentation is essentially random, whereasenzymatic fragmentation using restriction enzymes is sequence-specificand repeatable. Thus restriction digestion is a preferred method forfragmenting gDNA in a sample.

If both a test and a reference sample are being assayed, once thesamples have been fragmented they are then labelled with distinguishabledyes.

To give useful results in an array CGH method, the array must containprobes that match the fragmented genomic DNA. Every differentfragmentation of a genome will give different hybridisable sequences,and so there will be a different optimum set of probes for each of thefragmentations. Thus the best array for analsing a fragmented genomewill depend on the precise fragmentation method that was used. Theoptimisation of probe sets for use in array CGH is well known in the artand optimisation is a routine practice.

For a specific restriction enzyme, in silico digestion can show thefragments that will be produced. This information can be used to designa set of probes that are hybridisable to the restriction fragments andthat cover the genome to the desired degree. Moreover, it can be used todesign a set of probes that will offer an appropriate level of coverage.Probe design may also involve standard techniques, such as ensuring thatprobes are essentially unique within a target genome (i.e. that theyhave essentially no cross-hybridsation potential). If specific regionsare of interest then probes may be focused on these regions e.g. onsubtelomeres, on specific chromosomes, on specific genes, etc. Probedesign may also be restricted by the number of probes that can beincluded on the chosen array platform.

To provide enough material for hybridisation in array CGH in situationswhere the sample is limited, the DNA samples can be subjected toamplification. For example, reference 8 established the efficacy ofwhole genome amplification (WGA) approaches for achieving this goal.

First Probe Set

The first probe set functions as a CGH array and thus contains aplurality of hybridisation probes substantially complementary to aplurality of target nucleotide sequences in the genomic DNA sample. Thetarget nucleotide sequences may, for example, contain specific genes or,be from a chromosomal region suspected of being present at increased ordecreased copy number in cells of interest, e.g., tumour cells.

An array of such target sequences could represent locations that sampleeither continuously, or at discrete points, any desired portion of agenome, including, but not limited to, an entire genome, a singlechromosome, or a portion of a chromosome. The number of targetnucleotide sequences and the complexity of the nucleic acids in eachwould determine the density of sampling.

Preferably the first probe set will comprise ten or more hybridisationprobes per chromosome, i.e., 20, 30, 40, 50, 100, 150, 200, 250, 300,500, 1000, 2000, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000,45000, 50000 or more.

Preferably the hybridisation probes of the first probe set are between50 to 70 nucleotides in length, i.e., 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70. More preferably thehybridisation probes of the second probe set are 60 nucleotides inlength. The probes of the first probe set may all be the same length aseach other, or they may be different lengths.

Second Probe Set

The second probe set functions to track the chromosome and determine ifLOH has occurred. In a preferred embodiment the second probe setfunctions as a SNP array and comprises one or more pair(s) ofhybridisation probes for a SNP position, wherein the pair(s) of probesdiffer in sequence such that a nucleic acid target present in the samplecan differentially hybridise to the two probes depending on thenucleotide at the SNP position.

The inventors have empirically tested a large number of probes followingthe methodology described above and found that SNP probes suitable foruse under standard array CGH conditions are generally between 50-70nucleotides in length, e.g. 60 nucleotides in length.

The probes of the second probe set may all be the same length as eachother, or they may be different lengths. The probes will have a Tm ofbetween 65-75° C., i.e. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75° C.Preferably the Tm will be 72° C.

In addition the probes may contain a non-hybridising linker regionand/or a second destabilizing mutation. The probes may also comprise alocked nucleic acid (LNA) and/or a peptide nucleic acid (PNA).

As discussed above, a SNP is a DNA sequence variation occurring when asingle nucleotide —A, T, C, or G— in the genome (or other sharedsequence) differs across a population. SNP arrays rely on differentialhybridisation to distinguish between these differences and thehybridisation probes are designed to perfectly match the differentpolymorphisms of the SNP. Preferably the first probe of the pair isdesigned to perfectly match one polymorphism and the second probe of thepair matches the second polymorphism.

Preferably the invention uses SNPs with only two alleles. Morepreferably the invention uses SNPs with an allelic frequency of 40-60%,e.g. 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59 or 60%. Most preferably the SNP has an allelic frequency of 50%.

In a preferred embodiment of the invention, one of the pair ofhybridisation probes is designed to perfectly match one polymorphism andthe second probe is designed to perfectly match the second polymorphism.Preferably the pair of hybridisation probes is accompanied by a thirdand fourth probe which act as controls and correspond to the third andfourth bases respectively. For example, if the SNP has two alleles, Tand G, the first probe be designed to bind to the T allele, the secondprobe will be designed to bind to the G allele and the third and fourthprobes will be designed to bind to the same sequence containing C or Ainstead, thus acting as controls. The different probes for a single SNPdiffer in sequence from each other. If there is a single nucleotidedifferent between two probes for a single SNP, this is preferably not atthe 3′ terminal nucleotide of the probes. More preferably, the probesare identical at their 3′ terminal nucleotides. They may also beidentical at their 5′ terminal nucleotides. Thus they may differ insequence at 1 or more internal nucleotides.

Preferably, a probe's nucleotide at the SNP position is not its 3′terminal nucleotide. More preferably a probe's nucleotide at the SNPposition is not a terminal nucleotide, i.e. preferably a probe'snucleotide at the SNP position is an internal nucleotide. Preferably thetwo probes in a pair of hybridisation probes differ in sequence at atleast one internal nucleotide.

Preferably differential hybridisation of a probe to different allelesdoes not arise due to a mismatch at the 3′ terminal nucleotide. Morepreferably differential hybridisation of a probe to different allelesdoes not arise due to a mismatch at a terminal nucleotide i.e.preferably differential hybridisation of a probe to different allelesarises due to a mismatch at an internal nucleotide of the probe(s).

If the sample comprises a SNP which is homozygous then hybridisation ofthe sample will result in a signal which is higher for one of the pairof probes, i.e. the true match probe, compared to the other. A SNP whichis heterozygous will give approximately the same signal for both of thepair. This is summarised in the FIG. 2. The SNPs can then be tracked toshow regions of the chromosome that display Loss of heterozygosity(LOH).

LOH can be as a consequence of UPD (isodisomy) or as deletion. Theintegration of the array CGH data, which detects deletions in thechromosome, can be used to distinguish if LOH is caused by chromosomaldeletion or isodisomy. If chromosomal deletion has not occurred andstretches of SNPs which are homozygous as opposed to heterozygous aredetected, this leads to the conclusion that LOH has occurred due toisodisomy.

Preferably the second probe set will comprise multiple pair(s) ofhybridisation probes for different SNP residues. For example the secondprobe set may comprise 10, 20, 50, 100, 250, 500, 1000, 2000, 3000,5000, 10000, 15000 or more pairs of hybridisation probes.

Any given SNP in the human genome is represented in two complementaryDNA strands: a sense strand and an antisense strand. An A base in onestrand is paired with a T in the other, and a C is paired a G in theother. Thus to locate the nucleotide at any particular SNP residue it ispossible to analyse either strand and to infer the complementarynucleotide. The invention can look at the sense strand or the antisensestrand for any SNP. Where the invention includes two probes fordifferentially hybridising to two SNP targets it is usual that both ofthese should hybridise to the same target strand (i.e. both to the sensestrand or both to the antisense strand).

The invention may not involve a step of actually determining thenucleotide at a particular SNP position, but may instead merely identifydifferential hybridisation to one of two probes, but this hybridisationresult inherently reveals the nucleotide at the relevant positionbecause the probes have known sequence and differentiation capability.

Preferably, hybridisation of a genomic DNA sample to a probe is notidentified using a chain extension method, i.e. a method by which one ormore nucleotides are added to the 3′ terminal nucleotide of a probe.Preferably detection of hybridisation of genomic DNA sample to a probeis carried out without the addition of any further nucleotides to theprobe. Preferably, differential hybridisation of a genomic DNA sample toprobes is identified solely by observing hybridisation.

Preferably, label will not be incorporated into a probe during or afterhybridisation.

Preferably the second probe set will comprise five or more hybridisationprobes per chromosome, i.e., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300 or more. Thestatistical significance of the results will improve with an increasenumber of pairs of hybridisation probes. For example, if the secondprobe set comprises two pairs of probes per chromosome and the testsample is shown to be homozygous for both SNPs then there is a 25%chance that the homozygosity occurred by chance. If, on the other hand,the second probe set comprises 10 pairs of probes per chromosome and thetest sample is shown to be homozygous for all ten SNPs then there is a˜0.1% chance that the homozygosity occurred by chance.

The first and second probe set may comprise a different number of probesper chromosome, e.g. more, less or the same. For example the first probeset may comprise 10 pairs per chromosome and the second probe set maycomprise 50. Any combination is included in the invention.

Preferably the hybridisation probes of the second probe set are between50 to 70 nucleotides in length, i.e., 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70. More preferably thehybridisation probes of the second probe set are 60 nucleotides inlength.

The hybridisation probes on an array of the invention can bepre-synthesised before being applied to the array, or may be prepared onthe array in situ (e.g. by inkjet printing, by light-directed synthesis,etc.).

The skilled person will appreciate that as the second probe setfunctions to track the chromosome and determine if LOH has occurred itmay function as a SNP array, as described above, or may function todetect INDELs, VNTRs or transposons.

INDELs are short deletions or insertions that are only one or a few basepairs long. Instead of or in addition to targeting SNPs, the secondprobe set may include probes which are designed to designed to detectINDELs instead of SNPs. In this embodiment, one of the pair ofhybridisation probes in the second set is designed to hybridise to DNAthat contains the insertion and the second of the pair of hybridisationprobesin the second set hybridises to the empty site in the DNA, ordeletion. INDELs can be repetitive short insertions or deletions (morecommonly known as variable numbers of tandem repeats, VNTRs, or simplesequence repeats, SSRs, or microsatellites, etc.) and so in thisembodiment there may be more than one probe pair. An INDEL probe set maycomprise a series of probes that are designed to hybridise to DNAcontaining no insertion, one, two, three or more insertions.

VNTRs are tandem repeats present in the genome. Instead of or inaddition to targeting SNPs, the second probe set may comprise probesthat are designed to detect VNTRs. Allelic pairs comprise VNTRs withdifferent numbers of the repeated sequence. In this embodiment one ofthe set of hybridisation probes in the second set is designed tohybridise to DNA that contains one allele of the VNTR and a second ofthe set of hybridisation probes in the second set hybridides to DNA thatcontains another allele, which contains a different number of repeats.As VNTRs are variable repetitive short repeats with a range of differentnumbers of repeats, in this embodiment there may be more than one probepair to represent the number of alleles in the population. A VNTR probeset may be designed such that it comprises a set of probes that aredesigned to hybridise to DNA containing no repeat, one, two, three ormore repeats.

Transposons are movable genetic elements present in the genome. Insteadof or in addition to targeting SNPs, the second probe set may compriseprobes which are designed to detect transposons inserted in the genome.In this embodiment, one of the set of hybridisation probes in the secondset would be designed to hybridise to DNA without the presence of thetransposon. Other probes of the set of hybridisation probes in thesecond set would bind to DNA consisting of the transposon and thegenomic DNA transposon insertion site. The chimeric probes may comprisepartly of DNA complementary to the target genomic DNA and partlycomplementary to the transposon DNA.

The invention can be used with all such sequence variations e.g. SNPs,INDELs, VNTRs, and/or transposons (but in some places, for brevity, thetext may refer only to SNPs).

Nucleic Acid Array Materials

A nucleic acid array is a plurality of hybridisation probes immobilizedon a solid surface to which target nucleic nucleotide sequences can behybridised. This format permits a sample to be contacted simultaneouslywith the immobilised probes in a single reaction compartment.

The preparation and use of nucleic acid arrays, and methods for analsingthe hybridisation results obtained from them, are standard in the art.Preferred detection methods for analsing hybridisation results arefluorescence-based. For analysis typically specialist software which iswell known in the art will be used to detect the copy number variation.For example software available from OGT, Cytosure Interpret, AgilentGenome Workbench and BlueGnome BlueFuse Multi.

As described above, the present invention combines the advantages ofarray CGH and SNP arrays into a single assay format and thereforeprovides information on copy number changes in a given subject's DNA andprovides information about the nucleotide present at one or more SNP.The combination of this information allows a user to determine if LOH,if detected, has occurred as a result of isodisomy or chromosomaldeletion. The SNPs (or other variations) can then be tracked to showregions of the chromosome that display LOH. LOH can be as a consequenceof UPD (isodisomy) or as deletion. The integration of the array CGH datawhich detects deletions in the software can be used with the dataobtained from the SNP probes to distinguish if LOH is caused bychromosomal deletion or isodisomy.

The hybridisation probes used in the invention are usually nucleic acidmolecules. The hybridisation probes on an array will generally be atleast 30 nucleotides long (e.g. >40 nt, >50 nt, >60 nt, >70 nt, >80 nt,etc.). Thus the probes may be oligonucleotides (e.g. 40-80 nucleotideslong per probe), although it is also possible to use longer probes e.g.BAC DNA, PCR amplification products, etc.

The probes may be attached to the array non-covalently or, preferably,covalently.

Methods for immobilising nucleic acids onto array surfaces are wellknown in the art. Various methods for attaching nucleic acids tosurfaces in a hybridisable format are known e.g. attachment via linkers,to a matrix on a surface, to a gel on a surface, etc. The best-knownmethod is the photolithographic masking method used by Affymetrix for insitu synthesis of nucleotide probes on a glass surface, butelectrochemical in situ synthesis methods are also known, as are inkjetdeposition methods. References 9 and 10 review current methods, and alsoexperimental designs, which are appropriate to the invention.

Bead-based arrays may be used.

The probes can be attached by a 5′ terminal residue, by a 3′ terminalresidue, or by an internal residue. This choice may affect the detectiontechnique and vice versa.

Various materials can be used as the solid support in arrays e.g. aplastic material or, preferably, a glass.

Hybridisation probes are typically arranged in discrete patches, andeach patch can have an area of less than 10^(X) m², where X is selectedfrom −4, −5, −6, −7, −8, −9, −10, −11, −12, etc . Microarrays with patchsizes in the order of 10 μm×10 μm (i.e. 10⁻¹⁰ m²) are readily preparedusing current technology. Small patches can improve detectionsensitivity.

The centre-to-centre separation of patches is preferably less than10^(Y) m, where Y is selected from −2, −3, −4, −5, etc. Adjacent patchesmay abut or may overlap, but it is preferred that adjacent patches areseparated by a gap. Overlapping patches are not preferred.

Arrays preferably contain at least 10^(N) different analytical reagents,wherein N is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8 or more.Immobilisation of at least 10⁶ different oligonucleotides onto a singlesurface is well known in the field of microarrays. The 10^(N) differentreagents will typically be arranged in 10^(N) different patches.

Where the first probe set and second probe set are arranged in discretepatches on the array these patches may be interspersed with each other,or may be on different areas of the array. For example, the discretepatches of first probe set may be arranged in a distinct area to thoseof the second probe set. The same is true for the pairs of the secondprobe set, which may be in discrete patches next to each other orarranged separately on the array. Where pairs of the second probe setalso include control probes, these may be arranged in discrete patchesnext to the pairs or in separate areas of the array.

In preferred embodiments, the probes match regions of gDNA whichsubstantially lack superstructure associated with condensed metaphasechromosomes from which they are derived. The general nature of thepacking of DNA into eukaryotic chromosomes is well known to those ofskill in the art. Briefly, the superstructure of a eukaryotic chromosomecomprises many orders of complexity.

Probes for including on the array can be designed based on knowledge ofthe target sequences. In general, a probe will have a sequence selectedsuch that it is specific for a single target sequence i.e. probes thatcan hybridise to more than one target sequence are undesirable. Specifichybridisation in this way ensures that copy number polymorphism for aparticular target is directly related to the ratio obtained from thearray. Given the sequences of all targets, design algorithms can selectprobe sequences with the required specificity.

An array of the invention may include one or more replicates of aparticular immobilised nucleic acid and/or control nucleic acid e.g.duplicates, triplicates or quadruplicates. Replicates provideredundancy, provide intra-array controls, and facilitate inter-arraycomparisons.

Hybridisation Conditions

Preferably the hybridisation reactions of the invention are carried outusing standard hybridisation buffers known in the art. Preferably thehybridisation buffer is the hybridisation buffer available from Agilenttechnology (Catalogue number 5188-5380).

The hybridisation reactions are preferably carried out at a temperaturebetween 50° C. and 80° C., i.e. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79 or 80° C. Preferably the hybridisation reaction is carried out atabout 65° C., at 65±3° C., at 65±2° C., at 65±1° C., or ideally at 65°C.

Preferably all of first probe set and second probe set candifferentially hybridise to their respective nucleic acid targets underthe same hybridisation conditions.

Method for Simultaneously Performing Array CGH and SNP Array Analysis

References in this section to SNPs apply equally to INDELs, VNTRs, andtransposons.

As described above, the arrays of the invention are capable ofsimultaneously performing array CGH and SNP array analysis on a genomicDNA sample. The invention therefore provides a method for simultaneouslyperforming array CGH and SNP array analysis on a genomic DNA samplecomprising:

(a) contacting a nucleic acid array which comprises a first probe setand a second probe set with a genomic DNA sample, comprising a test andreference sample, under hybridisation conditions, wherein: (i) the firstprobe set, for the detection of copy number variation by array CGH,comprises a plurality of hybridisation probes substantiallycomplementary to a plurality of target nucleotide sequences in thenucleic acid sample; and (ii) and the second probe set comprises one ormore pair(s) of hybridisation probes for a SNP position, wherein thepair(s) of probes differ in sequence such that a nucleic acid targetpresent in the sample can differentially hybridise to the two probesdepending on the nucleotide at the SNP position;

(b) comparing the amount of test sample and reference sample hybridisedto the hybridisation probes of the first probe set;

(c) comparing the amount of test sample and reference sample hybridisedto the hybridisation probes of the second probe set; and

(d) using the data obtained in steps (b) and (c) to determine the copynumber of at least one locus; and at least one SNP in the genomic DNAsample.

In addition, the invention provides a method for distinguishing if lossof heterozygosity (LOH) at a locus is caused by chromosomal deletion orisodisomy comprising: (i) simultaneously performing array CGH and SNParray analysis on a genomic DNA sample according to a method of theinvention; (ii) using the data obtained from step (d) of part (i) todistinguishing if loss of heterozygosity (LOH) at a locus is caused bychromosomal deletion or isodisomy; wherein, if all of the SNPs locatedon a particular chromosome or region of a chromosome are identified ashomozygous and there is no indication of copy number variation in thesame region, then it is likely that the LOH is a consequence ofuniparental isodisomy; if the SNPs located in a particular chromosome orregion of a chromosome are not all homozygous and there is an indicationof copy number variation in that region, then it is likely that the LOHis a consequence of the chromosomal deletion and not from UPD.

The second probe set of the invention will usually comprise multiplepair(s) of hybridisation probes for different SNP residues. In themethod of the invention, if all of the SNPs located on a particularchromosome or on a particular portion of a chromosome are shown to behomozygous and there is no indication from the CGH data that achromosome deletion has occurred, then it is likely that the LOH is aconsequence of UPD (isodisomy). Alternatively, if out of the SNPslocated on a chromosome some are shown to be homozygous and there is anindication from the CGH data that a chromosome deletion has occurred,then it is likely that the LOH is a consequence of the chromosomaldeletion and not from UPD.

Therefore, there are three possible results obtained from the assays ofthe invention: 1) If SNP probes indicate LOH but the array CGH probesindicate a copy number variation (CNV) then it is likely that there is achromosomal deletion in the sample DNA; 2) if the SNPs probes indicateLOH but array CGH probes do not indicate CNV then it is likely that thesample DNA being tested contains isodisomy; and 3) if the SNP array doesnot indicate LOH and the array CGH probes do not indicate CNV, then itis likely that there is neither a chromosomal deletion nor isodisomy. Asdescribed above, the number of pairs of hybridisation probes in thesecond probe set affects the statistical significance of the dataobtained.

These methods will generally look at autosomal chromosomes.

The arrays of the invention can also be used to detect heterodisomy bytesting the parental DNA and the use of the SNP probes to track theparental origin of the DNA in the patient DNA.

Preferably the genomic DNA sample tested using the method of theinvention is a human DNA sample.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%. Where necessary, the term “about” can be omitted.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to “hybridisation” typically refer to specific hybridisation,and exclude non-specific hybridisation. Specific hybridisation can occurunder experimental conditions chosen, using techniques well known in theart, to ensure that the majority of stable interactions between probeand target are where the probe and target have at least 90% sequenceidentity. The hybridisation conditions can be used to aid the design ofprobes in arrays, such that probe sequences are not used if they havemore than 90% identity to other areas of the genome being analysed, tominimise cross-hybridisation. The stability of any particularprobe/target duplex analysed depend on the buffer/washing conditionsused. Stable duplexes are those that remain hybridised after washingsuch that they will contribute to the signal obtained for that probewhen reading the array.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Overview of array CGH.

FIG. 2: Signal intensity of different probes hybridised with homozygousor heterozygous samples.

FIGS. 3 & 4: Diagrams showing an ideogram of the results from anIsodisomy 8(FIG. 3) and an Isodisomy 15 (FIG. 4) sample using SNP andCGH probes. The arrows show an aberration (marked “A”) and the % ofhomozygous SNPs on a chromosome (“%”), which is indicated as a darkshading on a semicircular annulus.

FIGS. 5 & 6: Diagrams showing an ideogram of the results from anIsodisomy 8 (FIG. 5) and an Isodisomy 15 (FIG. 6) sample using indelprobes.

MODES FOR CARRYING OUT THE INVENTION Use of SNP and CGH Probes to DetectUPD

Array Fabrication A combined array comprising (i) ˜37,116oligonucleotide SNP probes (both allele probes, triplicate) to detect6,186 SNPs and (ii) ˜137,100 CGH probes was fabricated using ink jet insitu synthesis technology (see ref. 11) and was supplied by AgilentTechnologies Inc.

Labelling

The test DNA and reference DNA were labelled using the CytoSurelabelling kit (OGT catalogue number 020020). Briefly, the DNAs aredigested using Alul and Rsal for 2 hours at 37° C. Following digestionand denaturation of the enzyme by heating at 65° C. for 20 mins, 10 μlrandom primers and 10 μl reaction buffer were added and the mixincubated at 94° C. for 3 minutes. Following 5 minutes on ice, 10 μl ofnucleotide mix was pipetted into the mix. 1 μl of Cy3-dCTP was thenadded to the test DNA mix and 1 μl of Cy5-dCTP was added to thereference DNA mix. The reaction was started with the addition of 1 μl ofexo-free Klenow and incubation at 37° C. for 2 hours. The enzyme wasdeactivated by incubation at 65° C. for 10 minutes.

The labelled DNA was cleaned up using CytoSure purification columns (OGT020020). The columns were prepared by a spin at 2000 g for 1 minute. Themixes were each pipetted onto a column and the columns spun at 2000 gfor 1 minute. The labelled DNA was collected.

Hybridisation

DNA was prepared for hybridisation by mixing together the labelledsample and reference DNA, then adding Cotl, Blocking buffer (AgilentTechnologies Inc) and 2× high rpm hybridisation buffer (AgilentTechnologies Inc). The mix was denatured at 94° C. for 3 minutes,followed by incubation at 37° C. for 30 minutes. The hybridisation wasset up by pipetting the mix onto an Agilent backing slide (AgilentTechnologies Inc) and creating a ‘sandwich’ with the Microarray slide.The hybridisation was carried out using the SureHyb cassettes (AgilentTechnologies Inc) and incubated at 65° C. for 40 hours at a rotation of20 rpm in a SureHyb oven (Agilent).

Washing and Scanning

The cassettes were disassembled under aCGH Wash buffer 1 (Agilent) andthe slides washed with aCGH Wash buffer 1 for 5 minutes at roomtemperature. A second wash was carried out in aCGH Wash buffer 2(Agilent) for 1 minute at 37° C. The slides were scanned using anAgilent Microarray scanner at 2 μm resolution at 100% PMT settingfollowing the manufacturer's recommendation.

Feature Extraction and Analysis

Cy3 and Cy5 intensity data from the SNP and aCGH probes was extractedfrom the image files using Agilent's feature extraction software.

Copy number variations (CNVs) were identified from the aCGH results byexamining the ratio of the signals (Cy5/Cy3) on the CGH probes. Theaberrations were then identified using a combination of Circular BinarySegmentation (CBS) and calling the segment as an aberration using twothresholds set at a value of log2(Cy5/Cy3) exceeding 0.6 in the case ofdeletions and below −0.3 in the case of gains.

SNPs were genotyped by examining the ratio of the Cy3 signal on the twoalleles' probes. The obtained signals were corrected using a correctionfactor for each individual SNP which had been ascertained from previousexperiments with known genotyped samples. A threshold was then appliedto call the SNP as homozygous (allele 1, AA), heterozygous (AB) orhomozygous (allele 2, BB). The thresholds used were as follows:homozygous (allele 1, AA) a ratio of Cy3 signal allele 1/Cy3 signalallele 2 exceeding 0.5; heterozygous (AB) a ratio of Cy3 signal allele1/Cy3 signal allele 2 between 0.45 and −0.45; homozygous (allele 2, BB)a ratio of Cy3 signal allele 1/Cy3 signal allele 2 below −0.5).

A small number of probes were been excluded by filtering if they failedto achieve a minimum signal or reproducibility between the 3 replicates.

Results Detection of Isodisomy 8 UPD

Column A in Table 1 shows the number of SNPs that were homozygous oneach chromosome in an Isodisomy 8 sample. Column A in Table 2 shows theposition of CNVs detected using the CNV probes in the same Isodisomy 8sample.

Most chromosomes have 50±2% of their SNPs that are homozygous. Allchromosomes except for Chromosome 8 and X have between 38%-58% of theirSNPs that are homozygous. 91% of SNPs on chromosome 8 are homozygous,which indicates that the arrays have detected a significant loss ofheterozygosity (LOH) at chromosome 8. The CNV probes have not detected alarge deletion on chromosome 8, so this indicates that the LOH is due toisodisomy 8. The X chromosome also indicates a LOH on chromosome X. Thisis because the sample is a male sample and therefore contains a single Xchromosome. The reference used was male. FIG. 3 shows an ideogram withthe results of the SNP probes and the CNV probes.

Detection of Isodisomy 15 UPD

Column B in Table 1 shows the number of SNPs that were homozygous oneach chromosome in an Isodisomy 15 sample. Column B in Table 2 shows theposition of CNVs detected using the CNV probes in the same Isodisomy 15sample.

Most chromosomes have 50±2% of their SNPs that are homozygous. Allchromosomes except for chromosome 15 and X have between 42-58% of SNPsthat are homozygous.

89% of SNPs on chromosome 15 are homozygous, indicating a significantLOH on chromosome 15. Examination of the CNV probes detected noaberrations (by examining the ratio of sample signal/reference signal).Therefore this suggests that the LOH was not due to a deletion and wasdue to isodisomy on chromosome 15. The other chromosome indicating LOHwas chromosome X. This is because the sample is a male sample andtherefore contains a single X chromosome. The reference used was male.FIG. 4 shows an ideogram with the results of the SNP probes and the CNVprobes.

Use of Indel and CGH Probes to Detect UPD Array Fabrication

A combined array comprising oligonucleotide indel probes (both alleleprobes, in triplicate) to detect 490 indels and ˜43,323 CGH probes wasfabricated using ink jet in situ synthesis as described above. Note morethan one probe type may be used to detect each indel.

Labelling The test DNA and reference DNA were labelled using theCytoSure labelling kit (OGT 020020). Briefly, the DNAs are denatured for20 mins with 10 μl random primers and 10 μl reaction buffer at 99° C.for 20mins. Following 5mins on ice 10 μl of nucleotide mix was pipettedinto the mix. 10 μl of Cy3-dCTP was then added to the test DNA mix and10 μl of Cy5-dCTP was added to the reference DNA mix. The reaction wasstarted with the addition of 10 μl of exo-free Klenow and incubation at37° C. for 2 hours. The enzyme was deactivated by incubation at 65° C.for 10 minutes.

Labelled DNA was cleaned up using CytoSure purification columns, asdescribed above.

Hybridisation, Washing and Scanning

Hybridisation, washing and scanning was performed for the indel/CGHarrays in the same way as described above for the SNP/CGH arrays.

Feature Extraction and Analysis

Cy3 and Cy5 intensity data from the Indel and aCGH probes were extractedand normalised from the image files in the same way as described abovefor the SNP/CGH arrays. CNVs were identified from the aCGH results inthe same way. Indels were genotyped by examining the ratio of the Cy3signal on the two alleles' probes in the same way as described above forSNP genotyping.

Results Detection of Isodisomy 8 UPD

Column C in Table 1 shows the number of indels that were calledhomozygous on each chromosome in an known whole chromosome 8 isodisomysample. Column C in Table 2 shows the position of CNVs detected usingCNV probes in the same isodisomy 8 sample.

Most chromosomes have 50±10% of their indels that are homozygous. Thelow figure for chromosome 21 is likely due to incomplete coverage ofthis chromosome by indel probes. The number of homozygous indels onchromosome 8 is 91%, which indicates that the arrays have detected asignificant LOH at chromosome 8. The CNV probes have not detected alarge deletion on chromosome 8, so this indicates that the LOH is due toisodisomy 8. The X chromosome also indicates a LOH on chromosome X. Thisis because the sample is a male sample and therefore contains a single Xchromosome. The reference used was male. FIG. 5 shows an ideogram withthe results of the indel probes.

Detection of Isodisomy 15 UPD

Column D in Table 1 shows the number of indels that were homozygous oneach chromosome in an Isodisomy 15 sample. Column D in Table 2 shows theposition of CNVs detected using the CNV probes in the same isodisomy 15sample.

Most chromosomes have 50±10% of their indels that are homozygous. Allchromosomes except for chromosome 15, 18, 21 and X have between 43-68%of indels that are homozygous. 89% of chromosome 15 indels arehomozygous, indicating a significant LOH on chromosome 15. The highfigures for chromosomes 18 and 21 are likely due to the low number ofindels on these chromosomes (24 and 12 respectively). Examination of theCNV probes on chromosome 15 detected no aberrations (by examining theratio of sample signal/reference signal). Therefore this suggests thatthe LOH was not due to a deletion and was due to isodisomy on chromosome15. The other chromosome indicating LOH was chromosome X. This isbecause the sample is a male sample and therefore contains a single Xchromosome. The reference used was male. FIG. 6 shows an ideogram withthe results of the indel probes.

Use of VNTRs and CGH Probes to Detect UPD

Array fabrication

One VNTR probe on chromosome 8 appeared to perform adequately in testexperiments. This probe bound to the VNTR designated r58192897 by dbSNP.A combined array comprising this VNTR probe (both alleles in triplicate)and ˜15,159 CGH probes was fabricated using ink jet in situ synthesistechnology as described above.

Labelling

The test DNA and reference DNA were labelled using the CytoSurelabelling kit (OGT 020020). Briefly, the DNAs are denatured for 20minswith 10 μl random primers and 10 μl reaction buffer at 99° C. for20mins. Following 5mins on ice 10 μl of nucleotide mix was pipetted intothe mix. 10 μl of Cy3-dCTP was then added to the test DNA mix and 10 μlof Cy5dCTP was added to the reference DNA mix. The reaction was startedwith the addition of 1 μl of exo-free Klenow and incubation at 37° C.for 2 hours. The enzyme was deactivated by incubation at 65° C. for 10minutes.

Subsequent steps of (i) cleaning labelled DNA, (ii) hybridisation, (iii)washing, (iv) scanning, (v) feature extraction, and (vi) data analysiswere all performed in the same way as described above for the SNP andindel arrays. VNTRs were genotyped by examining the ratio of the Cy3signal on the two alleles' probes in the same way as described above forand SNP and indel genotyping.

Detection of Isodisomy 8 UPD

The CGH probes indicate that there is no large deletion on chromosome 8,and the VNTR probe calls its allele as homozygous. A single CNV wasdetected using the CGH probes, namely a 0.79Mb loss on chromosome 14. Asthere is no large CNV detected on chromosome 8 the homozygous call forthe VNTR probe indicates that the combination of VNTR probes and aCGHprobes can be used to identify UPD.

It will be understood that the invention has been described by way ofexample only and modification of detail may be made without departingfrom the spirit and scope of the invention.

TABLE 1 % of SNPs on indicated chromosome which are homozygousChromosome A B C D 1 49% 43%  53%  50% 2 48% 47%  54%  64% 3 46% 46% 75%  64% 4 51% 45%  69%  47% 5 41% 45%  65%  59% 6 51% 47%  54%  56% 748% 45%  58%  68% 8 91% 49%  91%  55% 9 45% 52%  50%  58% 10 51% 46% 68%  52% 11 52% 48%  67%  63% 12 58% 53%  54%  47% 13 48% 56%  79%  59%14 52% 42%  56%  43% 15 40% 89%  56%  89% 16 44% 48%  57%  53% 17 51%42%  69%  54% 18 54% 51%  52%  79% 19 49% 50%  57%  57% 20 54% 45%  47% 53% 21 55% 53%  8%  83% 22 38% 58%  54%  54% X 88% 86% 100% 100%

TABLE 2 Aberrations detected by CNV probes (exceeding 0.25 Mb) Onlythose CNVs exceeding 0.25 Mb are shown Chro- mosome A B C D 1 2 Loss0.94 Mb loss  1.5 Mb gain 0.27 Mb 3 4 1.17 Mb 2.23 Mb gain loss 5 2.02Mb gain 1.34 Mb gain 6 0.28 Mb gain 7  0.3 Mb gain 8 1.75 Mb, 0.52 Mb,1.16 Mb gain 9 10 11 0.48 Mb gain 0.39 Mb gain 12 2.95 Mb gain 13 140.34 Mb gain, 0.67 Mb gain, 0.32 Mb gain 1.49 Mb gain 15 Gain 0.31 Mbgain Gain 0.75 Mb 0.75 Mb 16 17 Loss 0.36 Mb gain Loss 0.47 Mb 0.47 Mb18 19 20 21 22 0.69 Mb loss 0.65 Mb loss, 1.75 Mb gain X

1-14. (canceled)
 15. A method for distinguishing if loss ofheterozygosity (LOH) at a locus is caused by chromosomal deletion orisodisomy comprising: (A) simultaneously performing array CGH and SNParray analysis; array CGH and INDEL array analysis; array CGH and VNTRarray analysis; or array CGH and transposon array analysis on a genomicDNA sample comprising steps (a) to (d): (a) contacting a nucleic acidarray which comprises a first probe set and a second probe set with agenomic DNA sample, comprising a test and reference sample, underhybridisation conditions, wherein: (i) the first probe set, for thedetection of copy number variation by array CGH, comprises a pluralityof hybridisation probes substantially complementary to a plurality oftarget nucleotide sequences in the nucleic acid sample; and (ii) thesecond probe set comprises one or more pair(s) of hybridisation probescomprising a first allele probe and a second allele probe, wherein theprobes are 50-70 nucleotides in length comprising a linker sequence ofup to 30 nucleotides in length for one or more of a SNP position anINDEL position a VNTR position a transposon position wherein the pair(s)of probes differ in sequence such that a nucleic acid target present inthe sample can differentially hybridise to the two probes depending onthe nucleotide at the SNP position, the sequence at the INDEL positionthe number of tandem repeats at the VNTR position the presence orabsence of a transposon at the transposon position wherein a probe'snucleotide at the SNP position is not its 3′ terminal nucleotide; (b)comparing the amount of test sample and reference sample hybridised tothe hybridisation probes of the first probe set; (c) comparing theamount of test sample hybridized to the first allele probe and thesecond allele probe of the second probe set; and (d) using the dataobtained in steps (b) and (c) to determine both the copy number of atleast one locus and one or more of at least one SNP in the genomic DNAsample at least one INDEL in the genomic DNA sample at least one VNTR inthe genomic DNA sample at least one transposon in the genomic sample (B)using the data obtained from step (d) of part (A) to distinguish if lossof heterozygosity (LOH) at a locus is caused by chromosomal deletion orisodisomy; wherein, if substantially all of the SNPs, INDELs, VNTR, ortransposons located on a particular chromosome or region of a chromosomeare identified as homozygous and there is no indication of copy numbervariation in the same region, then it is likely that the LOH is aconsequence of uniparental isodisomy; if the SNPs, INDELs, VNTR, ortransposons located in a particular chromosome or region of a chromosomeare not all homozygous and there is an indication of copy numbervariation in that region, then it is likely that the LOH is aconsequence of the chromosomal deletion and not from uniparentalisodisomy.
 16. The method of claim 15, wherein the test and referencesamples present in the genomic DNA sample are each labelled with a labeldistinguishable from each other.
 17. A nucleic acid array that, whencontacted with a genomic DNA sample under hybridisation conditions can:(a) provide information about the sample relating to the copy number ofone or more loci in the genome; and (b) distinguish between differentalleles present at one or more SNP, INDEL, VNTR or transposon position,comprising a first probe set and a second probe set, wherein: (i) thefirst probe set, for the detection of copy number variation by arrayCGH, comprises a plurality of hybridisation probes substantiallycomplementary to a plurality of target nucleotide sequences in thenucleic acid sample; and (ii) the second probe set comprises one or morepair(s) of hybridisation probes for a SNP, INDEL, VNTR or transposonposition, wherein the pair(s) of probes differ in sequence such that anucleic acid target present in the sample can differentially hybridiseto the two probes depending on the nucleotide at the SNP INDEL, VNTR ortransposon position; wherein a probe's nucleotide at the SNP position isnot its 3′ terminal nucleotide.
 18. The nucleic acid array according toclaim 17, wherein the probes of the first probe set are each greaterthan 60 nucleotides long.
 19. The nucleic acid array according to claim17, wherein the one or more pair(s) of the second probe set are each 60nucleotides long.
 20. The nucleic acid array according to claim 17,wherein the first and second probe set are DNA.
 21. The nucleic acidarray according to claim 17, wherein the SNP residue has an allelicfrequency between 40% - 60%.